Extracellular Vesicles Derived from Three-Dimensional-Cultured Human Umbilical Cord Blood Mesenchymal Stem Cells Prevent Inflammation and Dedifferentiation in Pancreatic Islets

It is unclear whether extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) have a direct protective effect on pancreatic islets. In addition, whether culturing MSCs in three dimensions (3D) instead of a monolayer (2D) can induce changes in the cargo of EVs that facilitate the polarization of macrophages into an M2 phenotype has not been investigated. We sought to determine whether EVs from MSCs cultured in 3D can prevent inflammation and dedifferentiation in pancreatic islets and, if so, whether the protective effect is superior to that of EVs from 2D MSCs. Human umbilical cord blood- (hUCB-) MSCs cultured in 3D were optimized according to cell density, exposure to hypoxia, and cytokine treatment based on the ability of the hUCB-MSC-derived EVs to induce the M2 polarization of macrophages. Islets isolated from human islet amyloid polypeptide (hIAPP) heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs. EVs derived from 3D hUCB-MSCs had more abundant microRNAs involved in M2 polarization of macrophages and had an enhanced M2 polarization ability on macrophages, which was optimized when the 3D culture condition was 2.5 × 104 cells per spheroid without preconditioning with hypoxia and cytokine exposure. When islets isolated from hIAPP heterozygote transgenic mice were cultured in serum-deprived conditions with hUCB-MSC-derived EVs, the EVs derived from 3D hUCB-MSCs suppressed the expression of proinflammatory cytokines and caspase-1 in pancreatic islets and increased the proportion of M2-polarized islet-resident macrophages. They improved glucose-stimulated insulin secretion, reduced the expression of Oct4 and NGN3, and induced the expression of Pdx1 and FoxO1. The greater suppression of IL-1β, NLRP3 inflammasome, caspase-1, and Oct4 and induction of Pdx1 and FoxO1 were found in islets cultured with the EVs derived from 3D hUCB-MSCs. In conclusion, EVs derived from 3D hUCB-MSCs optimized for M2 polarization attenuated nonspecific inflammation and preserved β-cell identity of pancreatic islets.


Introduction
Intravenous injection of mesenchymal stem cells (MSCs) improves the function and survival of pancreatic islets and preserves β-cell identity in animal models of type 2 diabetes [1][2][3] and pancreatic islet transplantation [4][5][6]. An impor-tant mechanism of these benefits is the ability of MSCs to induce polarization of macrophages into the M2 subtype. In type 2 diabetes and primary islet graft failure after pancreatic islet transplantation, the progression of inflammatory reactions such as the increase in islet macrophage infiltration, the polarization of macrophages into the M1 subtype, and the inflammasome activation play an essential role in the progression of β-cell failure [7]. Clinical trials on the infusion of MSCs into patients with type 2 diabetes [8] and recipients of pancreatic islet autotransplantation have been conducted without severe adverse events [9].
The use of MSC-derived extracellular vesicles (EVs) offers a promising alternative to MSCs themselves by reproducing their biological function in delivering nucleic acids, proteins, and lipids to the local microenvironment of damaged cells or tissues [10,11]. Moreover, EVs could decrease safety concerns because they are nonimmunogenic and not likely to cause maldifferentiation [12,13], which is a clinical concern of therapies using MSCs. Interestingly, it has been suggested that EVs derived from MSCs, without additional effects from MSCs themselves or other components of their secretome, can improve pancreatic β-cell survival and insulin sensitivity in rodents with low-dose streptozotocin and high-fat diet-induced diabetes [14]. This indicates that the use of MSC-derived EVs could be a novel approach to delaying the progression of β-cell failure in type 2 diabetes and primary islet graft failure after pancreatic islet transplantation.
MSCs cultured using the three-dimensional method (3D MSCs), which better reflects an in vivo environment, consistently exhibit enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation [15]. Such benefits are mediated, at least in part, by EVs from MSCs [16]. In addition to the changes in gene expression profiles such as the expression of genes with cell stemness and migration ability in 3D MSCs, EV production increases with the three-dimensional culture method, and the resulting EVs can have a cargo profile that is different from 2D MSCs [17]. In a recent study, total RNA sequencing using next-generation sequencing platforms on human amnion-derived 2D and 3D MSCs revealed profound transcriptome changes, including enhanced secretion of C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 12 (CXCL12), and bone morphogenetic protein 2 (BMP2), which could contribute to a microenvironment favouring polarization of macrophages into the M2 phenotype [18]. However, a previous study conducted in an animal model of bleomycininduced lung fibrosis suggested that EVs produced from 3D MSCs did not demonstrate enhanced immunomodulatory properties compared with 2D MSC-derived EVs [19], indicating that optimization of 3D MSC-derived EVs in each disease model is required.
EVs from cytokine-preconditioned MSCs contain several microRNAs (miRs) that can induce macrophage polarization into the M2 subtype, in contrast to EVs from resting MSCs [20,21]. However, whether the cargo changes in EVs derived from 3D MSCs include an enhanced capacity for the polarization of macrophages into the M2 phenotype has not been investigated, and there has been no specific study that compared the benefits of EVs from either 2D or 3D MSCs on pancreatic islets. Although it has been suggested that intraperitoneal injection of 3D MSCs instead of 2D MSCs in a multiple low-dose streptozotocin-induced diabetes model better attenuates inflammatory processes in pancre-atic islets and improves glycemic control [22], the role of 3D MSC-derived EVs in this benefit, if any, has not been determined.
Therefore, we investigated whether EVs from MSCs cultured in 3D have direct protective effects against inflammation and dedifferentiation on pancreatic islets and, if so, whether the protective effects are superior to those of EVs from monolayer-cultured MSCs (2D MSCs).

Materials and Methods
2.1. Animals and Cell Culture. Mouse islets were isolated from heterozygous human islet amyloid polypeptide (hIAPP) transgenic (hIAPP +/-) 8-12-week-old FVB/N mice (Jackson Laboratory, Bar Harbor, ME, USA). The Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute approved all animal experimental protocols in this study. Human umbilical cord blood-(hUCB-) MSCs were isolated according to a reported method [23]. Umbilical cord blood (UCB) units obtained from full-term deliveries were collected from the unborn placenta with the informed consent of the mothers. The human UCB-MSC isolation procedure was approved by the Institutional Review Board of Samsung Medical Center (IRB No. SMC 2019-11-026), and all participants provided informed consent for the use of the umbilical cord in this experimental study. The human monocyte cell line THP-1 was purchased from the Korean Cell Bank (Seoul, Korea) and maintained in complete RPMI 1640 media.

Mouse Islet Isolation and Culture.
Mouse islets were isolated from 8-12-week-old hIAPP +/mice as described previously [24]. Briefly, 0.8 mg/mL collagenase P (Roche, Basel, Switzerland) in Hanks' balanced saline solution (HBSS; Sigma-Aldrich) was infused into the common bile duct for mouse pancreas digestion. Islets were purified from the digested pancreas using a Human PanColl (PAN-Biotech GmbH, Am Gewerbepark, Aidenbach, Germany) gradient and washed several times with HBSS (Sigma-Aldrich). Purified hIAPP +/mouse islets were cultured in RPMI 1640 (Gibco) containing 10,000 U/mL penicillin-streptomycin (Gibco) and cultured at 37°C in a fully humidified 5% CO 2 atmosphere.  Table 1). For the microRNA real-time PCR assay, total RNA was extracted from 30 μg EVs using a Total Exosome RNA and Protein Isolation Kit (Thermo Fisher Scientific). The relative expression (ΔΔCt) of microRNA suggested to induce polarization of macrophages into M1 (miR-127-3p and miR-155-5p) and M2 (miR-34a-5p and miR-146a-5p) phenotypes which were determined using a Mir-X™ miRNA qRT-PCR 3 Stem Cells International TB Green® Kit (Takara), according to manufacturer's instructions. These microRNAs were chosen based on a previous study that used exosomes from proinflammatory cytokine-stimulated adipose MSCs [20]. The entire sequence of the mature microRNA can be used as the microRNAspecific 5 ′ primer.

Glucose-Induced Insulin Secretion (GSIS) Test. A GSIS
test was performed to measure islet functionality as described previously [24]. Islets cultured with or without hUCB-MSC-derived EVs were cultured in a KRBB solution containing low-(60 mg/dL) or high-concentration glucose (300 mg/dL). Twenty islets were used for each condition. After incubating for 60 min at 37°C, the supernatant was collected for measuring insulin concentration by ultrasensitive insulin enzyme-linked immunosorbent assay (ELISA) (ALPCO, Salem, NH, USA).

Enzyme-Linked Immunosorbent Assay and Cytokine
Array. To determine the expression level of cytokines, mouse islets (80 islets per well) were seeded onto a 6-well ultralow attachment plate (Corning, NY, USA). The culture medium was collected at 48 h without cellular debris. The concentrations of TNF-α, IL-1β, IL-6, and HMGB1 were quantified with an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. A cytokine array was performed using a Proteome Profiler Human XL Cytokine Array Kit (R&D Systems) following the manufacturer's instructions.
2.12. Cytokine Profiling Array. A cytokine array was performed using a Proteome Profiler Human XL Cytokine Array Kit (R&D Systems) following the manufacturer's instructions. For cytokine arrays, the membrane was blocked in buffer and incubated with 100 μg of EVs overnight at 4°C on a rocking platform shaker. Then, the membrane was incubated with a detection antibody cocktail for 1 h on a shaker. After washing, the membrane was incubated with streptavidin-HRP. Finally, Chemi Reagent mix was spread on the membrane, and an autoradiograph was obtained. Pixel densities were analyzed using NIH ImageJ medical imaging software.

Statistical Methods.
Results are reported as mean ± standard deviation. Statistical analysis was performed using GraphPad Software (GraphPad, San Diego, CA, USA). Continuous variables were compared using one-way analysis of variance (ANOVA) or the Mann-Whitney U test, as appropriate. P values < 0.05 were considered significant.

Characterization of EVs Derived from Umbilical Cord
Blood MSCs. To confirm the identity of the isolated hUCB-MSCs, surface expression of CD90, CD44, CD73, and CD105 and the nonexpression of the negative marker cocktail (CD45, CD34, CD14, CD11b, CD19, and HLA-DR) were confirmed using flow cytometry (Figure 1(a)). After conditioned medium was obtained during 2D and 3D cultures of hUCB-MSCs, hUCB-MSC-derived EVs were isolated from 4 Stem Cells International each group using a polymer-based precipitation method. The shape of each EV was observed using TEM. The cupshaped morphology of the isolated EVs was confirmed in both 2D and 3D hUCB-MSC-derived EVs (Figure 1(b)).
There was no significant change in the size distribution of EVs according to the MSC culture method (Figure 1(c)). When the expression of EV markers was examined using western blotting, CD63 and TSG101 were expressed, and the ER and Golgi markers CANX and GM130 were not expressed (Figure 1(d) and Supplementary Figure S1).   Figure S2a). Compared to the macrophages treated with 2D hUCB-MSC-derived EVs with and without cytokines (IFN-γ and TNF-α, each 40 ng/mL), the M2 polarization marker CD206 was upregulated in the macrophages treated with 3D hUCB-MSC-derived EVs. When the macrophages were treated with 3D hUCB-MSC-derived EVs, the expression of CD206 was higher when the seeding density of hUCB-MSC was 25 K cells per spheroid than for 2.5 K cells per spheroid. When macrophages were treated with 3D hUCB-MSC-derived EVs, the expression of CD206 was significantly higher in macrophages cultured with 25 K 3D hUCB-MSC-derived EVs than with 2.5 K 3D hUCB-MSCderived EVs (Figure 2(b)). Consistent findings were observed when M2 polarization markers (CD206 and CD163) of THP-1 monocytes were compared among groups (Supplementary Figure S3).

hUCB-MSC-Derived EVs
To evaluate whether the additional preconditioning of hUCB-MSCs, such as exposure to hypoxia and cytokine, further potentiates the M2 polarization ability of EVs, we obtained EVs from 3D cultures of hUCB-MSCs grown under hypoxia or cytokines (TNF-α and IFN-γ, each 40 ng/ mL) at a seeding density of 25 × 10 3 cells per spheroid ( Figure  2(c) and Supplementary Figure  S2b). Preconditioning with cytokines did not further increase the M2 polarization ability of 3D hUCB-MSC-derived EVs. When cytokine secretion of the macrophages cultured in the presence of 2D and 3D hUCB-MSC-derived EVs was 6 Stem Cells International measured by ELISA, the IL-10 and TGF-β levels were significantly increased in macrophages cultured in the presence of 25 K 3D hUCB-MSC-derived EVs (Figure 2(d)).
A recent study suggested that EVs from cytokinepreconditioned MSCs contain several miRs that can induce macrophage polarization into the M2 subtype, in contrast to the EVs from resting MSCs [20,21]. Thus, miRs in EVs isolated after preconditioning with IFN-γ and TNF-α in 2D-and 3D-cultured hUCB-MSC-derived EVs were quantified and compared with miRs in EVs isolated from 2D cultures without cytokine stimulation. When the cargoes of 2D and 3D (25 K) hUCB-MSC-derived EVs were analyzed, the level of miR-127-3p, a miR involved in M1 polarization [20], was decreased in 25 K 3D hUCB-MSC-derived EVs compared to 2.5 K and 2D hUCB-MSC EVs, resulting in a similar level to that in cytokine-treated 2D hUCB-MSC EVs (Figure 3(a)). There was no difference in the level of miR-155-5p between 25 K 3D hUCB-MSC-derived EVs and 2D hUCB-MSC-derived EVs. The level of miR-155-5p was increased in the 2.5 K 3D hUCB-MSC EVs ( Figure 3(a)). In 25 K 3D hUCB-MSC-derived EVs, the levels of miR-34a-5p and miR-146a-5p, miRs involved in M2 polarization [20], were significantly greater than those in unstimulated 2D, cytokine-stimulated 2D, and other 3D hUCB-MSC-derived EVs. A significantly higher level of miR-34a-5p was observed in 2.5 K 3D hUCB-MSC-derived EVs than in unstimulated and cytokine-stimulated 2D hUCB-MSC-derived EVs (Figure 3(a)). Based on these results, 25 K 3D hUCB-MSC-derived EVs without exposure to hypoxia and cytokines were used for further experiments.
A comparison of the protein expression profiles of 2D MSC-derived EVs and 3D MSC-derived EVs revealed that cytokines related to angiogenesis and inflammation, such as IL-6, MCP-1, MIC-1, IL-11, G-CSF, CCL20, and IL-27, were expressed in higher quantities in 3D MSC-derived EVs than in 2D MSC-derived EVs (Figure 3(b)). Significance of difference was determined using one-way ANOVA with Tukey's posttest. (b) Proteomic analysis of selected human cytokines from 2D and 3D hUCB-MSC-derived EVs. The error bars represent the standard deviation of measurements in two separate sample runs (n = 4). Significance of difference was determined using two-way ANOVA with Tukey's posttest. Columns, mean; bars, SD. * p < 0:05, * * p < 0:005, * * * p < 0:0005, and * * * * p < 0:0001.  8 Stem Cells International hUCB-MSC-derived EVs could inhibit nonspecific inflammation and β-cell dysfunction provoked by serum deprivation, islets isolated from hIAPP +/mice were cultured in media supplemented with BSA without FBS (BSA group), media supplemented with FBS (FBS group), and media supplemented with BSA without FBS but with EVs derived from 2D hUCB-MSCs (BSA+2D EV group) or 3D hUCB-MSCs (BSA+3D EV group) or cocultured with 2D hUCB-MSCs (BSA+2D MSC group) or 3D hUCB-MSCs (BSA+3D MSC group) in media supplemented with BSA without FBS. The mRNA transcription levels of IL-1β, IL-18, NLRP3, caspase-1, TNF-α, IL-6, and HMGB1 in the six groups were measured using quantitative real-time PCR (Figure 4(a)). The transcription levels of IL-1β, IL-18, NLRP3, caspase-1, TNF-α, IL-6, and HMGB1 were all significantly lower in the BSA+3D EV group than in the BSA group. The transcription levels of TNF-α, IL-18, IL-6, and HMGB1 but not IL-1β, NLRP3, and caspase-1 were also significantly lower in the BSA+2D EV group than in the BSA group. The transcription levels of TNF-α, IL-1β, IL-18, NLRP3, IL-6, and caspase-1 were lower in the BSA+3D EV group than in the BSA+2D EV group (Figure 4(a)). We then evaluated the effect of hUCB-MSC-derived EVs on GSIS in hIAPP +/islets, as shown in Figure 4(b). High-glucose (300 mg/dL)-stimulated insulin secretion islet cells from 3D hUCB-MSCderived EVs over a one-hour static incubation period were more numerous than in the BSA group. At least in part, the decrease in the transcription levels of IL-18, NLRP3, caspase-1, TNF-α, IL-6, and HMGB1 in the BSA+3D MSC group was reproducible in the BSA+3D EV group. The decrease in medium concentrations of TNF-α, IL-1β, and HMGB1 in the BSA+3D MSC group and the BSA+3D EV group was comparable, and the decrease in medium concentration of IL-6 in the BSA+3D MSC group was partly reproducible in the BSA+3D EV group (Figure 4(c)). The decrease in transcription levels of TNF-α and IL-6 in the BSA+2D MSC group was partly reproducible in the BSA+2D EV group. The decrease in medium concentration of TNF-α and in part of the IL-6 in the BSA+2D MSC group was reproducible in the BSA+2D EV group. The decrease in medium concentration of IL-1β in the BSA+2D MSC group was not reproducible in the BSA+2D EV group (Figure 4(c)).

hUCB-MSC-Derived EVs
To determine whether these findings are consistent in the setting of interaction among pancreatic islets, macrophages, and EVs from MSCs, we cocultured pancreatic islets from hIAPP +/mice and freshly isolated pancreatic macrophages using a transwell system, with or without medium supplementation of 2D or 3D hUCB-MSC-derived EVs. After 48 hours of coculture, the total RNA of the pancreatic islets was extracted and real-time RT-PCR was performed. The transcription levels of IL-1β, IL-18, caspase-1, TNF-α, IL-6, and HMGB1 were downregulated in the islets cocultured with pancreatic macrophages in the presence of 3D hUCB-MSC-derived EVs (Figure 5(a)). High-glucose (300 mg/dL)-stimulated insulin secretion over a 1 h static incubation period in islet cells from 3D hUCB-MSCderived EVs was greater than in the control group or the 2D hUCB-MSC-derived EV group (Figure 5(b)). To evaluate the hUCB-MSC-derived EV-mediated M2 polarization, cocultured macrophages were stained with CD80 and CD206. The proportion of CD80-CD206+ cells was significantly greater in the 3D hUCB-MSC-derived EV groups than in the control and 2D hUCB-MSC-derived EV groups,  Columns, mean; bars, SD. * p < 0:05, * * p < 0:005, * * * p < 0:0005, and * * * * p < 0:0001. † p < 0:05, † † p < 0:005, † † † p < 0:0005, and † † † † p < 0:0001 compared to the BSA group by one-way ANOVA and Tukey's posttest. 9 Stem Cells International suggesting that 3D hUCB-MSC-derived EVs promoted M2 polarization of macrophages in the setting of interaction among pancreatic islets, macrophages, and EVs from MSCs ( Figure 5(c)). When the concentrations of TGF-β and IL-10 in the supernatant were determined by ELISAs in this setting, both significantly increased in the presence of 3D hUCB-MSC-derived EVs compared to the control group. The concentration of IL-10 in the 3D hUCB-MSC derived EV group was significantly higher than that of the 2D hUCB-MSC derived EV group (Figure 5(d)).
To confirm whether the inhibition of nonspecific inflammation caused by EVs derived from hUCB-MSCs was associated with the M1/M2 polarization of islet-resident macrophages, the proportion of ARG1 + and CD80 + cells in the islets of each group was compared. Although the proportion of ARG1 + cells in the islets of the BSA+2D EV group was not different from that of the BSA and FBS groups, the proportion of ARG1 + cells in the islets of the BSA+3D EV group was higher than in the other groups. Although the proportion of CD80 + cells in the islets of the BSA+2D EV group was not different from that of the FBS group, the proportion of ARG1 + cells in the islets of the BSA+3D EV group was lower than in the BSA, FBS, and BSA+2D EV groups (Figures 6(a) and 6(b)). The proportion of IL-1β + cells in the islets of both the BSA+3D EV and BSA +2D EV groups was lower than that of the BSA group. A more potent reduction in the proportion of IL-1β + cells in islets was observed in the BSA+3D EV group, which was similar to that of the FBS group (Figures 6(a) and 6(b)).

hUCB-MSC-Derived EVs Attenuate Dedifferentiation of hIAPP +/-Mouse Pancreatic Islets during Serum-Deprived
Culture. Metabolic stress-induced proinflammatory cytokines can induce the dedifferentiation of islet cells [25,26]. Therefore, the mRNA transcription levels of β-cell identity markers were compared among the four groups. The mRNA transcription levels of Oct4 and NGN3 in the BSA+2D EV and BSA+3D EV groups were significantly lower than those of the BSA group. In the BSA+3D EV group but not in the BSA+2D EV group, the mRNA transcription levels of Pdx1 and FoxO1 were significantly higher than those in the BSA group, with a significantly higher level in the BSA+3D EV group than in the BSA+2D EV group (Figure 7(a)). The protein expression of β-cell identity markers was evaluated using immunocytochemical staining. Similar to real-time RT-PCR assays, immunostaining showed that the expression levels of Pdx1 and FoxO1 were significantly higher in the BSA+3D EV group than in the BSA group. The proportion of FoxO1-positive cells was~3-fold higher in both the EVs (20 μg/mL) derived from the 3D hUCB-MSC and 2D hUCB-MSC groups than in the BSA group. The proportion of islet cells expressing Oct4 and NGN3 was downregulated in FBS, 2D hUCB-MSC-derived EVs, and 3D hUCB-MSCderived EVs. The proportion of islets expressing Oct4 was lower in the 3D hUCB-MSC-derived EVs than in the 2D hUCB-MSC-derived EVs.
In addition to alleviating the dedifferentiation of islets, the proportion of islets expressing cleaved caspase-3 was significantly lower in both the 3D hUCB-MSC-derived EV and 2D hUCB-MSC-derived EV groups than in the BSA group (Figures 7(d) and 7(e)), whereas there were no significant differences in the numbers of hIAPP oligomer-positive cells between the BSA and 2D or 3D hUCB-MSC-derived EV groups.

Discussion and Conclusions
In this study, miRs involved in the M2 polarization of macrophages were more abundant in EVs derived from 3D hUCB-MSCs than in 2D hUCB-MSC-and 3D hUCB-MSC-derived EVs that possessed an enhanced M2 polarization ability on THP-1 monocytes. The protective effects of the 3D hUCB-MSC-derived EVs on hIAPP heterozygote transgenic mouse islets were more potent than 2D hUCB-MSC-derived EVs in terms of reducing nonspecific inflammation and preserving β-cell identity, and this was associated with a higher proportion of islet-resident macrophages with M2 polarization markers. To the best of our knowledge, these results are the first evidence that 3D hUCB-MSC-derived EVs have a direct protective effect on hIAPP-expressing pancreatic islets, and the M2-polarizing ability of the EVs might be an important contributor to such protective effects.
The greater M2-polarizing ability of EVs derived from 3D hUCB-MSCs compared to those derived from 2D hUCB-MSCs is a novel finding of this study. Although a recent study revealed that human adipose mesenchymal stem cell-(AMSC-) derived EVs can directly induce the M2 polarization of macrophages through the direct effect of miRs related to M2 polarization, such as that of miR34a-5p and miR 146a-5p [20], it has not been determined if the levels of such miRs can be amplified in 3D cultures of hUCB-MSCs. In the optimized 3D culture conditions in this study, miR34a-5p and miR 146a-5p were more abundant in EVs derived from 3D hUCB-MSCs than in those derived from 2D hUCB-MSCs, even when the 2D MSCs were preconditioned by IFN-γ and TNF-α. In addition, miR127-3p was less abundant in 3D than in 2D hUCB-MSC-derived EVs. miR-34 inhibits the transcription of proinflammatory cytokines by targeting Notch1, and miR-146 targets NF-κB signaling mediators such as IRAK1 and TRAF6 to promote the expression of M2-associated genes [20,27,28]. These results would be relevant to the enhanced protective effects of 3D MSC-derived EVs on βcells because macrophages are associated with inflammation in islet cells and β-cell dysfunction [7]. The injection of hUCB-MSCs into a type 2 diabetes mouse model polarized M1 macrophages to M2 macrophages in islets [3], reduced apoptosis of β-cells, and increased PDX-1 and MafA expressions. The results of our study indicate that similar effects can be reproduced by 3D hUCB-MSC-derived EVs alone, without the help of cellular components.
Although the results of our study are in line with previous research suggesting the superior immunomodulatory  Figure 5: M2 polarization ability of hUCB-MSC-derived EVs in a coculture system of islets and pancreatic macrophages. In a transwell system, 50 islets and 1 × 10 6 macrophages were cultured under three conditions: islets with macrophages without hUCB-MSC-derived EVs (islet w/MΦ), islets with macrophages with 2D hUCB-MSC-derived EVs (islet w/MΦ+2D EVs), and islets with macrophages with 3D hUCB-MSC-derived EVs (islet w/MΦ+3D EVs). (a) Concentrations of IL-1β, IL-18, NLRP3 inflammasome, caspase-1, TNF-α, IL-6, and HMGB1in cocultured hIAPP +/islet cells treated with EV produced by 2D-or 3D-cultured hUCB-MSCs were measured using qRT-PCR. The error bars represent the standard deviation of measurements in three separate sample runs (n = 9). (b) Glucose-stimulated insulin secretion (GSIS) of hIAPP +/mouse islets in each group. GSIS was evaluated using ELISA at 48 h after statin incubation of islets in low (60 mg/dL) and high (300 mg/dL) glucose. The error bars represent the standard deviation of measurements in three separate sample runs (n = 6). (c) Flow cytometry analysis of cell surface molecules CD206 and CD80 on macrophages cultured in the presence of EVs. The error bars represent the standard deviation of measurements in three separate sample runs (n = 9). (d) Medium concentrations of TGF-β and IL-10 were measured by ELISA. The error bars represent the standard deviation of measurements in four separate sample runs (n = 9). Columns, mean; bars, SD. * p < 0:05, * * p < 0:005, * * * p < 0:0005, and * * * * p < 0:0001 by one-way ANOVA and Tukey's posttest. 11 Stem Cells International effects of 3D MSCs over 2D MSCs [15,16,18,22], some previous studies reported contradictory results [19,37]. In one study, T cell-suppressive abilities of MSCs were observed only in 2D MSCs and not in 3D MSCs; in that study, the T cell-suppressive abilities of 3D MSCs were partly restored by addition of a corticosteroid [37]. Another previous study      14 Stem Cells International compared the immunomodulatory potency of 2D MSCderived EVs and 3D MSC-derived EVs in vitro and then compared their anti-inflammatory and antifibrotic potentials in vivo using a bleomycin-induced lung fibrosis model [19]. In that study, the in vitro immunomodulatory potency of 2D MSC-derived EVs and 3D MSC-derived EVs was compared after IFN-γ stimulation. The T cell suppression ability in terms of indoleamine 2,3-dioxygenase activity after IFN-γ stimulation and macrophage phenotype in terms of phagocytosis activity were lower in 3D MSC experiments, indicating polarization into the M1 subtype, although they did not directly measure M1 or M2 markers on macrophages [19]. In our study, the M2-polarizing ability of 3D MSCs was optimized when 3D MSCs were generated with sufficient cell numbers, without hypoxia or cytokine stimulation. In 25 K 3D MSCs, cytokine stimulation reduced the M2-polarizing ability. Therefore, it is possible that the immunomodulatory properties of 3D MSC-derived EVs could be lost if 3D culture conditions are not optimized. We also used a different source of MSCs (human UCB-MSCs in the current study vs. human lung tissue-derived MSCs or bone marrow-derived MSCs in the previous study [19]) and different disease models (serum-deprived culture of isolated pancreatic islets and cocultivation of hIAPPproducing pancreatic islets, MSCs, and macrophages). In this context, it is reassuring that the uniform-sized 3D MSCs produced by nanopatterned culture plasticware prevented βcell death in a multiple low-dose streptozotocin-induced diabetes model [22], in which immune cell infiltration and pro-gressive loss of β-cells typically occurs. Whether 3D MSCderived EVs alone without the cellular component would have similar benefit should be explored in future research. An important translational potential in our study is the use of EVs derived from 3D hUCB-MSCs in the pretransplant cultures of isolated human islets before clinical islet transplantation. Our previous study showed that serum deprivation, as in pretransplant cultures in clinical intraportal islet transplantation to avoid the use of animal-derived materials, induces an inflammatory response in cultured hIAPP +/islets even without the prolonged culture with hyperglycemia [24]. This study hypothesized that the addition of EVs derived from 3D hUCB-MSCs during the pretransplant culture of islets could attenuate the inflammatory response and loss of β-cell identity caused by serum deprivation. In this study, 3D hUCB-MSC-derived EVs promoted the M2 polarization of islet-resident macrophages and reduced the inflammasome activation induced by serum deprivation in hIAPP heterozygote mouse islets. This resulted in improved islet viability and insulin response to glucose, attenuation of proinflammatory cytokine expression, and preservation of β-cell identity. In addition to the enhanced M2 polarization ability of 3D hUCB-MSCderived EVs, this therapeutic effect could result from differences in the cargo and the proteome profiles of EVs derived from 2D and 3D hUCB-MSCs, which include several angiogenesis-related cytokines.